Previous work has indicated that chicken and human neuronal tissue pre- mRNA encoding nonmuscle myosin heavy chain (MHC) II-B can be alternatively spliced, resulting in the insertion of 10 amino acids in loop 1 and/or 21 amino acids in loop 2 (Takahashi et al., J. Biol. Chem. 267, 17864, 1992). We have used the Baculovirus system to express a fragment of human myosin heavy chain II-B (MHC) (amino acids 1-1045) along with two pairs of myosin light chains (MLCs), MLC20 and MLC17. This fragment is an HMM equivalent of the noninserted myosin II-B isoform which is present in many human tissues and cells. We have also expressed the same HMM with an insert of 21 amino acids in loop 2 with and without an insert of 10 amino acids in loop 1, along with MLC17 and MLC20. In avians and mammals, these isoforms are only expressed in neuronal tissue, especially in the adult human cerebrum (Itoh and Adelstein, ibid 270, 14533, 1995). The size of expressed proteins was approximately 110 kDa. Both the inserted and noninserted expressed HMMs were soluble and bound reversibly to actin in an ATP-dependent manner. Both contained a 1:1:1 molar ratio of MHC:MLC20:MLC17. The results of an in vitro motility assay indicated that noninserted HMM propelled actin filament at a slightly higher rate than the inserted isoform.